User:Victoria Mock/Sandbox 1

GREEN FLUORESCENT PROTEIN: Superfolder Mutant
Though several GFP variants have been created with improved folding abilities, 'superfolder' GFP (PDB 2b3p) holds claim to being one of the fastest and best-folding mutants. In addition to the previously discovered ‘cycle-3’ and ‘enhanced GFP’ mutations, superfolder GFP contains six new mutations: Y39N, N105T, Y145F, I171V, A206V, and most notably, S30R. With a more resilient structure and two-fold greater fluorescence than wild-type GFP, the superfolder variant serves as a better reporter of fusion protein expression. 

Background
Green Fluorescent Protein (GFP) is a valuable tool in cellular and molecular biology, largely because of the self-fluorescing properties of its chromophore. This fluorescence permits the molecule to function as a protein fusion tag in bacterial cells. Wild-type GFP and many of its mutant variants tend to misfold when used as protein fusion tags, often due to poor folding of the bacterial proteins themselves, which then hinders GFP from assuming its proper conformation and causes it to aggregate and display weaker fluorescence. Consequently, a “robustly folded” GFP variant, called superfolder GFP, was developed to withstand any folding complications of the protein it was designed to tag.

The Discovery of Superfolder GFP
Prior to superfolder GFP, a less efficient folding reporter GFP was designed to contain the known cycle-3 and enhanced GFP mutations. Cycle-3 mutations F99S, M153T, and V163A were found to prevent problems with GFP aggregation, most likely due to decreased hydrophobicity on the protein surface. Enhanced GFP mutations S65T and F64L increased fluorescence by 35-fold as compared to the wild-type by altering local interactions in and around the chromophore. The additional amino acid mutations unique to superfolder GFP that confer its superior durability and folding kinetics are located within the protein's beta-can structure, away from the central chromophore. These mutations are Y39N, N105T, Y145F, I171V, A206V, and most notably, S30R. Collectively, all six superfolder mutations decrease folding interference, but by different means.

Structural Details: S30R Mutation
The S30R superfolder mutation is most crucial to folding robustness and kinetics. Wild-type GFP possesses a serine residue on beta strand S2 at position 30, which is mutated to arginine in superfolder GFP, changing the local conformation and interactions with four key surrounding residues. A five-membered ion-pair network of alternating acidic and basic charges is created by glu-32, arg-30, glu-17, arg-122, and glu-115. This system of residues is the ultimate source of superfolder GFP's increased stability, with arginine as the main director of the arrangement. Arginine's ability to participate in double salt bridges to glutamic acid or other arginine residues allows for the electrostatic interactions that eliminate aggregation issues and make this protein the fastest and best-folding variant.

Additional Mutations
The other residue mutation that contributes most to overall folding success is Y39N. This supplies an extra hydrogen bond that enhances structural and folding robustness. The Y145F and I171V mutations eliminate aggregation-prone intermediates from GFP’s normal folding pattern. Mutations N105T and A206V may help with thermodynamic stability and increasing the yield of expressed protein. Individually, A206V is suspected to prevent dimerization of the protein, explaining the superfolder variant’s monomeric structure and its unique crystal packing pattern.

Benefits of Superfolder GFP
Superfolder GFP is one of the fastest folding GFP variants yet to be revealed. Its accuracy in folding regardless of its surrounding proteins, combined with fluorescence twice that of wild-type GFP, makes it ideal for use in protein tagging. Exceptional folding qualities also convey the ability to refold well after treatment with chaotropic agents as well as resistance to the deleterious effects of random mutation.